The project consists of analyses of growth and differentiation in a preneoplastic lesion of the mouse mammary gland, the hyperplastic alveolar nodule. Cell-culture methodology is used to compare selected properties of these cells with the same properties of cells from normal (midpregnant) and neoplastic epithelium of the mouse mammary gland. The objective is to learn how differentiation of mammary parenchyma is altered as neoplasia progresses. The emphasis is on regulation of mammary function by exogenous factors, especially hormones and heterotypic cell types. Cell lines of preneoplastic cells and of tumors which arose within preneoplastic nodules have been established and characterized, both in vivo and in vitro. One key cell culture property which we are assaying is growth in suspension in methylcellulose and soft agar, since cells which grow in suspension are generally considered to be neoplastic and we planned to select such cells for further analysis. To date, essentially no growth of either preneoplastic or tumor cells in suspension has been noted. Efforts to determine why the cells do not grow are in progress. As a marker for mammary differentiation, we have chosen to assay for synthesis of the disaccharide lactose. A chromatographic method for assaying lactose in cell culture fluids has been devised. Monolayer cultures of normal midpregnant mammary cells do not make lactose even if stimulated with insulin, prolactin, and hydrocortisone. A series of experiments is being conducted in which the role of mesenchymal cell types in lactose production in culture is being assessed. We will then attempt to learn how lactose production becomes altered in the preneoplastic cells.